Risk factors for acquiring Acinetobacter baumannii infection in the intensive care unit: experience from a Moroccan hospital

Introduction. Acinetobacter species are non-fermenting and ubiquitous Gram-negative coccobacilli, which in recent years have become the leading cause of healthcare-associated infections worldwide. Our objective here was to study the epidemiology and risk factors associated with Acinetobacter baumannii infections in the intensive care unit (ICU). Methods. This retrospective case-control study was conducted collaboratively between the Medical Bacteriology Department and the two ICUs of the Military Hospital of Instruction Mohammed V-Rabat over a 3 month period. Results. We included 180 patients, of whom 60 had A. baumannii infection. We observed a male predominance in both matched groups, with a sex ratio of 1.6. The median age was 67 years [interquartile range (IQR) 59.5–77]. The median length of stay in the ICU before infection was 8.5 days (IQR 5–14). Multivariate logistic regression analysis identified the risk factors statistically associated with A. baumannii infection at the ICU level as follows: duration of invasive procedures >7 days [odds ratio (OR)=1.02], parenteral nutrition (OR=3.514), mechanical ventilation (OR=3.024), imipenem (OR=18.72), colistin (OR=5.645), probabilistic antibiotic therapy >4 days (OR=9.063) and neoplastic pathology (OR=5.727). Conclusion. Based on our results, it can be inferred that shortening the duration of stay in the resuscitation setting, implementing rational use of medical devices and optimizing antibiotic therapy could decrease the incidence of these infections.


InTRoDuCTIon
Acinetobacter is a ubiquitous, non-fermenting Gram-negative coccobacillus that has become the leading cause of healthcareassociated infections worldwide in recent years due to its ability to survive in the hospital environment and to acquire resistance to commonly used antibiotics.It has been implicated in a wide range of conditions including skin and soft tissue infections, pneumonia, osteomyelitis and bacteraemia.
The emergence of multi-drug-resistant Acinetobacter baumannii complicates the therapeutic process and contributes directly to the increase in mortality and indirectly to the increase in length of stay and hospitalization costs.In Moroccan intensive care units (ICUs), Acinetobacter species accounted for 24.85 % of all isolates and 31.5 % of all Gram-negative bacilli [1].A recently published study demonstrated that the clonal spread of A. baumannii clinical isolates was related to those isolated from the hospital environment in two Moroccan ICUs [2].The same research team also reported that clinical isolates of A. baumannii were more resistant to antiseptics and disinfectants than those from the environment [3].This species has also become a matter of great concern due to its extraordinary ability to acquire resistance to commonly used antibiotics.Polymyxins remain the last therapeutic option but the emergence of colistin-resistant A. baumannii isolates has been reported worldwide [4].Over the last decade, antibiotic resistance rates of A. baumannii strains have increased from 78.3 to 100 % for piperacillin/tazobactam, from 68.7 to 100 % for ceftazidime, from 31.4 to 87.7 % for imipenem, from 27.3 to 100 % for amikacin, and from 77.8 to 100 % for ciprofloxacin in Moroccan ICUs [1,5,6].
According to the literature, the recognized risk factors associated with A. baumannii infections are: invasive procedures, previous hospitalization, host factors, length of stay in the ICU and previous use of broad-spectrum antibiotics [4].These infections are associated with mortality ranging from 28.3 to 84.3 % in the ICU [5,7].Independent predictors of mortality vary from country to country and region to region, and may be related to ICU-acquired infections, ineffective empirical antibiotic therapy, antibiotic resistance, immunosuppression, septic shock, medical device use and steroid use [7][8][9][10].Local data are monocentric and fragmentary and sometimes without separation between services.It is in this context that we conducted this study with the objective of studying the epidemiology and risk factors associated with A. baumannii infections in the ICU of the Mohammed V Military Training Hospital.

Study design and study site
This is a retrospective case-control study conducted in collaboration between the medical bacteriology department and the two resuscitation departments of the Mohammed V military training hospital in Rabat over a 3 month period from June to September 2022.

Study population
The original population consisted of patients hospitalized in the two series of medical and surgical resuscitation departments of the Military Hospital Mohammed V Rabat and having a positive biological workup for diagnostic purposes.
For our case-control study, 60 cases and 120 controls were randomly matched in a 1 : 2 ratio.Cases and controls were selected by including patients aged over 15 years, hospitalized in the two ICUs of the same facility and during the same study period.We excluded patients aged under 15 years of age, duplicates and patients whose records were lost.
Cases were defined as any patient infected with A. baumannii according to the Centers for Disease Control and Prevention criteria.Infection was considered acquired in the ICU if it occurred 48 h after admission to the ICU.Controls were defined as patients hospitalized in the ICU without A. baumannii infections.Clinical and microbiological data were collected from patient records and the laboratory information system using a questionnaire with the following variables: age, gender, patient's origin, underlying comorbidities, invasive procedures, previous probabilistic antibiotic therapy, notion of prior hospitalization up to 1 year before inclusion, length of stay in an ICU setting, bacterial co-infection, microbiological data (antibiogram, sampling site, type of sampling) and evolution.

microbiological method
Identification of bacterial isolates was based on culture, morphological and biochemical identification characteristics.Biochemical identification was performed using API20NE ready-to-use galleries (bioMérieux).
Antibiotic susceptibility was studied using the Mueller Hilton agar diffusion method by using OXOID-type antibiotic discs and interpreted according to the recommendations of EUCAST 2022.The interpretation was performed using the Adagio Biorad system.Quality control of the antibiotic susceptibility test was performed with Escherichia coli strain ATCC 25922.
For colistin, determination of the MIC was performed by the microdilution method using the sensititre COL* plate (Thermo Scientific Wellwash; RRID:SCR_020569) from an 18-24 h culture.Interpretation of the results was performed according to EUCAST 2022 recommendations.

Statistical methods
Data were entered into Microsoft Excel, RRID:SCR_016137.Statistical analysis was performed using Jamovi version 2.3.4.Quantitative variables were expressed as mean±sd or median (interquartile range -IQR) and qualitative variables as number and percentage.Comparison of qualitative variables was performed by Pearson's and Fisher's chi-square exact tests, and comparison of quantitative variables was performed by Student's t-test and Mann-Whitney U-test according to the normality of the distribution.Multivariate analysis was performed using a logistic regression model.
Measures of association were calculated with 95 % confidence intervals by setting a threshold of statistical significance at P≤0.05.

Patient characteristics
We included 180 patients meeting the eligibility criteria, 60 of whom had A. baumannii infection.We noted a male predominance in both matched groups with a sex ratio of 1.6.The median age of our population was 67 years (IQR 59.5-77).The median length of stay in the ICU before infection was 8.5 days (IQR 5-14).
Among 420 patients hospitalized in the ICU during the study period, 60 developed A. baumannii infection.The incidence of A. baumannii care-associated infections was 14.2 per 100 patients.
Study of the resistance of A. baumannii isolates to antibiotics showed resistance rates of 100 % for ticarcillin, piperacillin, piperacillin-tazobactam, ticarcillin-clavulanic acid, cefepime, ceftazidime, gentamicin, amikacin, imipeneme and ciprofloxacin.Tetracylin and minocyclin were resistant in 94 and 74% of isolates respectively.All isolates of A. baumannii were susceptible to colistin.Following the results of the resistance study, we deduced that all our isolates were represented by extremely drug-resistant A. baumannii (XDR) (Fig. 1).

Risk factors for A. baumannii infection in the resuscitation setting
Variables found to be statistically significant in univariate analysis were: disorders of consciousness, duration of invasive procedures >7 days, previous use of central venous catheters, arterial catheters, mechanical ventilation, parenteral nutrition, co-infection, probabilistic antibiotic therapy, probabilistic antibiotic therapy >4 days, previous use of colistin, imipenem, documented antibiotic therapy, clinical course, length of stay in the ICU and length of stay in the ICU >14 days (Table 2).3).

Risk factors associated with mortality
Regarding the mortality rate, 30 % of patients infected with A. baumannii died during their hospitalization compared to 16 % in the control groups (P<0.029).
Univariate analysis suggested that the following were risk factors for mortality: A. baumannii infection, duration of probabilistic antibiotic therapy <4 days, chronic smoking and neoplastic pathology (Table 4).
However, multivariate logistic regression with the variable selection method considering those risk factors with a P˂0.05, those with a P<0.3 and also those recognized in the literature associated with mortality by A. baumannii infection showed that A. baumannii infection, sepsis and probabilistic antibiotic therapy <4 days were significant independent predictors (P<0.005)(Table 5).

DISCuSSIon
A. baumanni is one of the most common opportunistic agents causing healthcare-associated infections, especially in the ICU setting.In the absence of incontrovertible data on A. baumannii infections, it is difficult to accurately estimate the incidence and thus the prevalence of multidrug-resistant bacterial infections including multidrug-resistant A. baumannii.In our study, the incidence of A. baumannii infections in the ICU setting was 14.2 %.Our rate is higher than that reported by Jean et al. (8.4 %), and that reported in India (10%) and in Mexico in cancer patients (4.6 %).This could be explained by differences in the application of hygiene and infection control measures, in particular hand hygiene practices and decontamination of the hospital environment [11].
In our study, independent risk factors for A. baumannii infection can be classified into three categories: those related to increased length of stay in the ICU, those related to the use of invasive medical devices (use of central venous catheters or mechanical ventilation and procedures ≥7 days), and those related to previous drug treatment (probabilistic antibiotic therapy >4 days, imipenem, colistin).
According to the literature, risk factors vary between countries and regions.The most frequently reported risk factors were previous exposure to carbapenems and previous antibiotic therapy [12][13][14][15][16][17].
A meta-analysis of 18 studies identified risk factors for multidrug-resistant bacterial infections.Male gender, exposure to surgery, central venous catheter, mechanical ventilation, previous antibiotic therapy and prolonged hospitalization were considered as risk factors for multidrug-resistant infections [18].Following our multivariate analysis, mechanical ventilation, use of probabilistic antibiotic therapy >4 days, parenteral nutrition, duration of invasive procedures >7 days, colistin, imipenem and neoplastic pathologies were independent risk factors.Our case differs from previous studies in case definitions, anatomical site of infection, antibiotic treatment protocols and antibiotic resistance profile.
Mechanical ventilation is the major factor in the emergence of nosocomial pneumonia caused by multidrug-resistant A. baumannii.In China, a retrospective incidence study showed that the use of mechanical ventilation led to a 2.5-fold higher risk of developing a nosomial infection with multidrug-resistant bacteria [19].In Greece, a prospective study showed that mechanical ventilation was a risk factor independently associated with the occurrence of healthcare-associated infections with multidrug-resistant bacteria.In our study, multivariate analysis showed that the use of mechanical ventilation increased the risk of acquiring healthcare-associated infections with multi-drug-resistant A. baumannii by three times compared to control patients [20].Intravascular devices are essential to modern medicine in the management of patients but their presence is not without infectious risk.This explains why A. baumannii isolates are frequent in respiratory infections and bacteraemia.
Our data also showed that imipenem and colistin increased the risk of A. baumannii infection by 18 and five times, respectively.
Prior exposure to antibiotics has been identified as a risk factor for multidrug-resistant bacterial infection in several studies [21,22].This highlights the importance of rational use of antibiotics.In our study, probabilistic antibiotic therapy longer than 4 days increased the risk of acquiring A. baumannii infection nine times compared to the control group.
Furthermore, our data showed that neoplastic pathology increased the risk of A. baumannii infection by 5.7 times in the cases compared with in the control group.
According to our study the risk of death is almost two times higher in patients infected with A. baumannii than in controls.
The literature is conflicting regarding the impact of bacterial resistance on mortality.A very high mortality rate has been reported in patients infected with extended-spectrum beta-lactamase (ESBL) enterobacteria, multidrug-resistant A. baumannii and multidrug-resistant Pseudomonas aeruginosa, while some studies have reported similar mortality rates for cases and controls.Several studies over the past two decades have demonstrated that inappropriate initial antimicrobial therapy is an independent risk factor for mortality in patients with multidrug-resistant bacteria [26][27][28][29][30][31][32][33][34][35][36].
Septic shock was statistically associated with two-fold greater mortality.Previous studies have shown that septic shock remains the leading cause of death in patients with multi-drug-resistant bacterial infection in the ICU and is a risk factor for mortality with inappropriate initial antimicrobial therapy [37].Our data also showed that probabilistic antibiotic therapy of less than 4 days increases the risk of mortality by 2.07.Being infected with multidrug-resistant A. baumannii has been shown to be associated with a 2.43-fold increased risk of mortality.

ConCLuSIon
Our data show that reducing the duration of ICU stay, rational use of medical devices and optimizing the initial empirical antibiotic therapy could significantly reduce the incidence of these infections.Patients with A. baumannii infection and septic shock have a poor prognosis.A local antibiogram database is needed to better monitor the evolution of bacterial resistance in our hospital.

Funding Information
The author(s) received no specific grant from any funding agency.

Author response to reviewers to Version 1
Risk factors for acquiring Acinetobacter baumanniiinfection in the intensive care unit: experience from a Moroccan hospital.

Author's response to editor
We would like to thank all the reviewers for their valuable comments and suggestions.Please find below our point by point responses to the comments.
These are the answers to the Editor comments and suggestions: • Please upload figures as separate, high resolution, editable files.Acceptable file types are PDF, GIF, TIFF, EPS, JPEG, PNG, SVG, and PPT.Please ensure the legends are in the main manuscript.
o Answer: thank you for this comment.It has been modified.

Author's response to reviewer 1
We would like to thank all the reviewers for their valuable comments and suggestions.Please find below our point by point responses to the comments.
These are the answers to the reviewer 1 comments and suggestions: • Please rate the manuscript for methodological rigour o Answer: thank you for this comment.it has been taken in consideration.

Author's response to reviewer 2
We would like to thank all the reviewers for their valuable comments and suggestions.Please find below our point by point responses to the comments.
These are the answers to the reviewer 2 comments and suggestions: • In abstract introduction, colon should be placed instead of semi-colon.
o Answer: thank you for this comment.It has been corrected.• Some light should be focused on extensively drug resistant (XDR) and pan drug resistant (PDR) A. baumanii strains also in this study.
o Answer: thank you for this comment.All our strains of Acinetobacter baumannii were extremely drug-resistant.Line no. 162-164.
• Role of healthcare workers can also be discussed as one mode of transmission of multi drug resistant strains.Other risk factors which could be considered or mentioned as hospital infection control policies like hand hygiene, regular cleaning of ICUs, entry and exit of attendants.
o Answer: thank you for this comment.We didn't study these factors in our study, but in the discussion we mentioned that they are factors that enable the spread of multi-drug resistant bacteria in the hospital setting.Line no. 206-208.  1 is showing resistance profile so in legend also it should be resistance profile and not susceptibility profile.8. Please give space between "care and" in line no.187.9. Reference required of meta-analysis study mentioned in line nos.233-236.10.There is no data revealed about the immune status of patients as it could also be a risk factor for causing infections among ICU patients.11.Some light should be focused on extensively drug resistant (XDR) and pan drug resistant (PDR) A. baumanii strains also in this study.12. Role of healthcare workers can also be discussed as one mode of transmission of multi drug resistant strains.Other risk factors which could be considered or mentioned as hospital infection control policies like hand hygiene, regular cleaning of ICUs, entry and exit of attendants.13.In conclusion, it should be suggested that a local antibiogram database is required for the hospital.

Please rate the manuscript for methodological rigour Good
Please

Comments:
The manuscript discusses about an important pathogen, Acinetobacter baumannii, which is more commonly a multi-drug resistant organism.Growth of this pathogen in an ICU setting and risk factors associated with it, is a relevant issue that needs to be highlighted and discussed.The authors have presented the manuscript well, the results and tables are nicely presented.Discussion part is also well-written, although there are a few grammatical errors here and there.Overall, the manuscript is good and can be accepted.

AQ5 Peer review history VERSIon 2 Editor
recommendation and comments https://doi.org/10.1099/acmi.0.000637.v2.1 © 2023 Tolman L. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Lindsey Tolman; University at Albany, UNITED STATES Date report received: 28 August 2023 Recommendation: Accept Comments: Reviewer comments have been sufficiently addressed in the revised manuscript.

Table 1 .
The distribution of bacterial co-infection in patients with A. baumannii infection

Table 3 .
Results of multivariate logistic regression analysis of risk factors associated with Acinetobacter baumannii infection

Table 4 .
Results of univariate analysis of risk factors for mortality in patients infected with Acinetobacter baumannii

Table 5 .
Results of multivariate logistic regression analysis of risk factors for mortality in patients infected with Acinetobacter baumannii

•
It should be mentioned as multidrug resistant and not as multi-resistant o Answer: thank you for this comment.It has been corrected.Line no.68.•Once the organism has been named fully, afterwards it should be mentioned as A. baumanii only.oAnswer:thank you for this comment.It has been corrected.•Lineno.88 should be in continuation with above paragraph i.e. line no.87.oAnswer:thank you for this comment.It has been corrected.lineno.83-86•Inline number 71, Intensive care unit should be written as ICU also in bracket as it has been used as ICU in line no.87.Similarly at other places also it should be mentioned as ICU.o Answer: thank you for this comment.It has been corrected.•Inline no.133,Microsoftexcel should not be in bracket.oAnswer:thank you for this comment.It has been corrected.Line no.131.•Figure 1 is showing resistance profile so in legend also it should be resistance profile and not susceptibility profile.oAnswer:thank you for this comment.It has been corrected.Line no.165.•Pleasegive space between "care and" in line no.187.oAnswer:thank you for this comment.It has been corrected.•Referencerequired of meta-analysis study mentioned in line nos.233-236.oAnswer:thank you for this comment.It has been added.Line no.220.•There is no data revealed about the immune status of patients as it could also be a risk factor for causing infections among ICU patients.oAnswer:thank you for your comment, in our study we studied subjacent pathologies such as; diabetes, cancer, hypertension... see table2, 4 © 2023 Singh M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.This is a well written manuscript and makes an original contribution.However, few changes are recommended to be considered by the author.1.In abstract introduction, colon should be placed instead of semi-colon.2. It should be mentioned as multidrug resistant and not as multi-resistant (e.g. in line no.69). 3. Once the organism has been named fully, afterwards it should be as A. baumanii only.4. Line no. 88 should be in continuation with above paragraph i.e. line no.87. 5.In line number 71, Intensive care unit should be written as ICU also in bracket as it has been used as ICU in line no.87.Similarly at other places also it should be mentioned as ICU. 6.In line no.133, Microsoft excel should not be in bracket.7. Figure • In conclusion, it should be suggested that a local antibiogram database is required for the hospital o Answer: thank you for this comment.It has been taken in consideration.Comments:The reviewers have highlighted minor concerns with the work presented.Please ensure that you address their comments.Reviewer 2 recommendation and commentshttps://doi.org/10.1099/acmi.0.000637.v1.3 rate the quality of the presentation and structure of the manuscript GoodTo what extent are the conclusions supported by the data?Strongly supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?YesReviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000637.v1.4 © 2023 Gupta N.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.